CRL- LAL Times - May 2, - 1999 VALIDATION OF BACTERIAL ENDOTOXINS TEST METHODS

Vol. 6, No. 2 May, 1999

EDITOR'S COMMENTS

This issue begins a two-part series on BET validation from the manager's viewpoint. The first part
discusses planning and execution of BET validation for an end product. The second part will
address the contents and conclusions in the final report. Sound science and good documentation
are the keys to a successful validation project. Dr. James F. Cooper

Dr. James F. Cooper

VALIDATION OF BACTERIAL ENDOTOXINS TEST METHODS

James F. Cooper

Bacterial endotoxins test (BET) method validation is used to document that a BET procedure will detect
endotoxin in a specific drug formula or device extract without interference. This discussion addresses the
components of BET validation.

Overview. The critical elements in a BET validation are the formulations of the test material, LAL reagent and
CSE. LAL reagents of FDA-approved suppliers differ in composition and manner in which they are processed.

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Differences include metal cation, protein, buffer and surfactant content. Test materials that are water extracts,
low-concentration solutions and pH neutral exhibit a similar compatibility profile in the various reagents, i.e.,
little or no detectable differences in interference properties. However, more complicated drug formulae, which
have buffers, metal chelators and components that aggregate endotoxin, may react differently in each reagent.
Therefore, the required validation for each LAL vendor is justified for most drug formulations.

Objective. The goal of a BET validation is to find a valid, compatible range (concentration or dilution) for
routine LAL testing of the material in question. The following is a summary of steps required to validate a BET
method in today's regulatory environment and technically advanced state of LAL testing.

STEPS IN BET METHOD VALIDATION

State the objective.

Describe the product.

Determine the endotoxin limit.

Define validation experiments.

Conduct physical measurements.

Make a 2-method compatibility profile.

Define validation conditions.

Conduct a 3-lot validation.

Prepare the validation report.

Creation of a new BET method should follow a validation protocol which encompasses the basic validation
steps and satisfies regulatory and pharmacopeial requirements.

Description of Test Material. The components of the test material must be described in detail.

A description of a pharmaceutical includes the generic and trade names, potency and fill volumes of the
product, category of use, product codes, complete formula, pH range and other product specifications. A
method for rehydrating a lyophilized product is needed to determine the resultant potency.

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CRL- LAL Times - May 2, - 1999 VALIDATION OF BACTERIAL ENDOTOXINS TEST METHODS

A description of a medical device includes the name, contents, chemical composition, directions for use,
and fluid pathway or parts of the device that contact tissue.

Endotoxin Limit (EL). The tolerance limit and maximum human dose per hour, as described in directions for
use, are required to calculate the endotoxin limit by the K/M formula. It is prudent to check clinical publications
to determine if there are dosage patterns that exceed the labeled dose range. Pharmacopeial limits are used,
if available. The USP 24, which becomes effective in 2000, will contain the most comprehensive list of
endotoxin limits with over 650 entries. If differences occur between pharmacopeia, it is prudent to use the
most conservative one applicable in the marketing area.

The category of the test material may fall in one of the unique EL classes such as a radioactive drug, infusion
solution or intrathecal material. The EL for combination drugs, (multivitamins, etc.) are best determined by
using the volume of the dose in the K/M formula. (1)

Validation Experiments. The experiments needed to document non-interference must be anticipated at the
outset to assure the task is done completely and expeditiously. The principal experiment for a drug product is
an interference screen with positive controls to find the dilution or concentration that is compatible with one or
more reagents. A measurement of pH is needed to determine if pH contributes to interference. Solubility may
be an issue for colloids, suspensions or drugs immiscible with water. Experimentation may be required to
devise a meaningful, reliable way to elute complex devices so that tissue sensitive parts or fluid pathways are
evaluated.

Influence of pH. A pH profile is an essential part of validation for products that are buffered or have a pH
range other than neutral. As an example, an antibacterial agent, having a pH range of 9 to 11, was diluted with
LRW as shown in Table 1. The pH was measured before and after mixing equal parts of each solution with
EndosafeÒ reagent. The results demonstrate the importance of dilution and use of buffered LAL for
neutralization. Although the pH of the mixture of LAL and sample (SPL) diluted to 1:10 was within the USP
allowable range, only concentrations £ 0.5 mg/mL allowed full recovery of the endotoxin spike because of
other interference mechanisms. Data must verify that the validated method neutralizes the most extreme pH,
such as pH 11 for this antibiotic (above).

Table 1: Antibiotic/LAL pH Profile

Dilution mg/mL SPL SPL/LAL
1:2 200 10.1 8.5
1:10 40 9.0 7.6
1:100 4 8.3 7.2
1:1000 0.4 7.5 7.0

Use of buffers and surface active agents in validated methods is discouraged for the following reasons: 1) LAL

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CRL- LAL Times - May 2, - 1999 VALIDATION OF BACTERIAL ENDOTOXINS TEST METHODS

reagents are extremely sensitive to pH, divalent cations and surface active agents; 2) the effect of these
agents varies with each batch of drug product and LAL reagent. 3) It is difficult to know whether an additive
has overcome inhibition or just enhanced the recovery of the endotoxin control. Reliance on LRW dilution to
resolve inhibition simplifies the validated method and avoids lot-to-lot variation in recovery. A pH-related
interference may be avoided by selecting a properly buffered LAL.

Of course, there are exceptions to the caution against buffers. For example, anticoagulant solutions containing
citrate cannot be tested without both magnesium replacement and neutralization. As previously discussed,
many raw materials present neutralization and dissolution problems that may require additives. (2)

Compatibility Profile. A compatibility profile is an extension of a preliminary screen for inhibition. The sole
purpose of this profile is to find the compatible concentration (CC) of the drug entity that doesn't inhibit or
enhance endotoxin recovery. The traditional way to find compatibility is to test a series of 2-fold dilutions of a
sample and identify the range where the Positive Product Control (PPC) is fully recovered. The 2 PPC in gel
clot methods only assures 50% recovery; a more informative method is a screen with at least 2 LAL methods
(gel clot and kinetic) to gain comprehensive knowledge about interference and discover how efficiently dilution
resolves interference. Kinetic turbidimetric analysis (KTA) is the most cost-effective choice of LAL method for a
compatibility profile.

The LAL-compatible concentration for most potent pharmacological agents is usually 5 mg/mL or less. Assays
of 2-fold dilutions in an 8-to-0.03 mg/mL series usually reveals the compatible range. (2) Sensitivity is not an
issue during preliminary tests because sensitivity may be adjusted after compatibility studies are analyzed. For
simplicity, one may select a of 0.0625 EU/mL for a gel-clot screen or a standard curve range of 0.05 to 5 EU/
mL for a KTA study.

A compatibility profile provides valuable information about the interference characteristics of a test
material. First, the profile reveals how efficiently dilution with LRW resolves interference conditions. Most
drugs require a 100-fold dilution to go from no recovery to ideal compatibility with LAL. Some require a 4 to 10-
fold dilution or greater to go from 50 to 100% recovery as seen in Table 2. Also, the presence of unsuspected
glucan contamination may be revealed by evaluating enhancement of PPCs in kinetic studies. Finally, the
profile may show that one LAL method is much more suitable than the others. The study in Table 2 was done
efficiently because two LAL methods were done with the same sample preparations and LAL reagent,
Endosafe®KTA.

Table 2. Compatibility Profile for a Contrast Media Drug with LAL*.

Potency (mg/mL) 120 60 30 15 7.5 3.7 1.8
2 PPC Recovery -- -- -- ++ ++ ++ ++
% PPC Recovery 6 25 59 81 91 103 105
*Endosafe® gel clot / KTA reagent

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CRL- LAL Times - May 2, - 1999 VALIDATION OF BACTERIAL ENDOTOXINS TEST METHODS

Define Validation Conditions. The compatible concentration (CC) or dilution is identified by analyzing the gel-
clot and kinetic results of the compatibility profile. The CC isn't the first 2 recovery or kinetic recovery over
50%. Rather, the CC is the highest, non-interfering test concentration that consistently recovers near 100% by
kinetic LAL and the 2 spike by gel clot.

When only gel clot data is collected, the ideal concentration is the 2nd or 3rd positive PPC. This precaution is
taken because the highest concentration with a positive result only assures 50% recovery. How does one
select the concentration for validation? A 5 mg/mL concentration of the drug in Table 2 is a defensible choice
of CC for validation because it is clearly within the compatible range, greater than the MVC and is a
convenient concentration to prepare.

The sensitivity of the test conditions in the profile must be calculated and compared to the endotoxin limit to
determine if there is adequate sensitivity. The Product Specific Sensitivity (PSS) is found by dividing the
lambda by the test concentration of a small volume parenteral, or by multiplying lambda by the dilution factor
for extracts.(1) If the PSS is not at least 4 times more stringent than the EL, sensitivity should be increased by
reducing lambda to provide a better margin of safety between the PSS and EL. Generally, the validated
concentration or dilution comes from the less dilute part of the compatible range, i.e., the range between the
compatible concentration (mg/mL) and the MVC for drugs, or between the compatible dilution and the MVD for
extracts and infusion solutions.

Validation of Three Lots. The Acceptance Criteria for initiating the final phase of validation are primarily
compatibility, sensitivity and pH. Validation of product compatibility with a specific LAL reagent by gel clot
requires a pharmacopeial method. The best choice for kinetic method is one described in FDA regulated use-
instructions from the LAL vendor. Acceptance criteria for a validated method include proof that the test
conditions achieved pH neutrality and that there was no significant difference between endotoxin detected in a
water control and in a product, as modified by dilution, extraction, etc. A validated method limits the maximum
concentration or least dilution of product that can be tested with a specific LAL reagent. It doesn't restrict a
change in lambda provided the reagent formula does not change.

The supplies, reagents and human resources required to produce and fully document a BET validation
are substantial. It is cost effective to validate multiple LAL methods at the same time because time-
consuming, costly aspects of validation, such as sample preparation and documentation, can be done
for two about as easily as for one.

Validation Report. The final report must fully document a description of the product and results of all
experiments during the preliminary testing and final validation experiments. The components of a validation
report will be discussed in the next issue of LAL Times.

References:
1. Cooper JF, BET calculations for multiple component parenterals, LAL Times, Vol. 5, No. 3, 1998. 2.

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CRL- LAL Times - May 2, - 1999 VALIDATION OF BACTERIAL ENDOTOXINS TEST METHODS

Cooper JF, LAL interference screening of inprocess materials and finished products, LAL Times, Vol. 5, No.
1, 1998.

Kenneth E. Avis, DSc., Professor Emeritus, 1918-1999

The parenteral drug industry was saddened by the passing of Ken Avis, my close friend and
mentor. Ken was our most visionary educator in the parenteral drug sciences. He founded the
Sterile Products Laboratory at the University of Tennessee (UT) College of Pharmacy which
became a model for post graduate training in the manufacture, processing and control of
parenteral medications. He held every major office in the PDA, received countless research
and teaching awards, and the FDA Commissioner's Special Citation in 1987. I had the good
fortunate to train with Ken as an undergraduate and later join the faculty at UT and work in his
Division. He provided endless encouragement and the opportunity to continue my development
work on LAL applications during the seventies.

Dr. Avis' research activities include a major contribution to LAL technology. Avis and Ludwig
published landmark papers on dry-heat inactivation of endotoxin as measured by LAL methods.
They designed a heating cell block with precise control of the heating rate and studied
inactivation kinetics of endotoxin in capillary tubes. They reported that the contrasting results of
earlier investigators was in part due to different but simultaneous rates of endotoxin
inactivation. They observed that a second-order model described inactivation between 250°
and 325° C, a finding that influenced BET requirements for dry-heat ovens. These studies
found that fillers in endotoxin standards potentiated thermal inactivation results. For this reason
Endotoxin IndicatorsÔ are made without filler.

Following are the citations for these noteworthy publications:

1. Validation of a heating cell for precisely controlled studies on the thermal destruction of
endotoxin in glass. J Parenteral Sci Technol, 42:9-14, 1988.

2. Recovery of endotoxin preparations from the surface of glass capillary tubes. J Parenteral
Sci Technol, 43:276-278, 1989.

3. Dry heat inactivation of endotoxin on the surface of glass. J Parenteral Sci Technol, 44:4-12,
1990.

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CRL- LAL Times - May 2, - 1999 VALIDATION OF BACTERIAL ENDOTOXINS TEST METHODS

CALENDAR

May 25-27 - LAL Workshop

presented by Charles River Endosafe, Ltd.,
at Stoke Rochford Hall, Lincolnshire, UK.
A lab and lecture series on current LAL topics.

June 2-5 - European Dialysis & Transplant Nurses Association, Berlin.

Presentation by Dr. Cooper:
"Endotoxin: Control, Analysis and Clinical Relevance

CHARLESTON ANNUAL LAL WORKSHOPS

June 23-26 - Gel Clot Workshop

June 28-July 1 - Quantitative Workshop

Contact: Frances Cooper for information
Phone: 843-795-7316
Fax: 843-795-7221

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